Therapeutic strategies that can deliver bioactive signals at different times during tissue formation are essential for the regeneration of complex tissues such as a mature vasculature. During normal wound healing, the events that lead to mature blood vessel formation results from a series of tightly regulated events, which occur sequentially as a result of environmental changes. This proposal focuses on the design, synthesis and in vitro testing of a non-viral gene delivery strategy that can deliver DNA in a temporally controlled fashion following environmental changes. In our approach, cationic polymer condensed DNA (polyplex) are covalently immobilized to biomaterials through matrix metalloproteinase (MMP) sensitive peptides that can be degraded following MMP addition. The peptides utilized to mediate polyplex immobilization will be designed so that they are cleaved by specific MMPs. Thus, DNA polyplexes encoding for different proteins can be immobilized to the biomaterial through peptides that can be degraded by different MMPs and their release, uptake and expression can be temporally controlled by the addition of different MMPs at different times. In vivo, the MMP expression profile is tightly regulated throughout the wound healing process with different MMPs being expressed at different times during tissue morphogenesis. The long term goal of the proposed research is to take advantage of this MMP expression profile during wound healing to deliver different pro-angiogenic proteins at different times to promote the formation of a mature vasculature and thus enhance the rate of wound healing. This proposal is divided into two aims. Aim 1 is the synthesis and characterization of triblock copolymers composed of three distinct blocks A, B and C, which can mediate DNA condensation into polyplexes, DNA polyplex immobilization and DNA polyplex release through specific MMPs. The A block will be composed of a MMP labile peptide, which can mediate immobilization through a terminal cysteine group and release through MMP degradation. The other two blocks, B and C, will be composed of poly(ethylene glycol) (PEG) and poly(ethylene imine) (PEI), which will be responsible for mediating DNA polyplex stabilization and DNA condensation. Peptide synthesis and amine/carboxylic acid chemistry will be used to synthesize the proposed ABC triblock copolymers. Aim 2 is to Induce cell triggered gene transfer by plating adhered cells on biomaterials that have DNA polyplexes covalently immobilized on their surface. Peptides that are degraded by specific MMPs will be used to immobilize the polyplexes and are expected to result in gene transfer only when the specific MMP is either added as a recombinant protein or released by stably transfected cells. Further, temporal control will be achieved by immobilizing polyplexes, encoding for different reporter genes via MMP labile peptides that are degraded by different MMPs. Thus, release of specific polyplexes can be controlled by adding specific MMPs at different times. PUBLIC HEALTH REVELANCE Angiogenesis, the formation of new blood vessels, represents a pressing clinical need for the treatment of ischemic wounds and is a major obstacle in the translation of tissue engineered constructs. One major limitation in the generation of mature blood vessels is the inability to deliver therapeutic molecules at the necessary times. This proposal aims to design a gene delivery strategy that can deliver DNA (the therapeutic) at the required times for angiogenesis to take places by using biologically regulated molecules to induce release at specific times during wound healing.